Isolation and sequence analysis of NBS–LRR disease resistance gene analogues from hop Humulus lupulus L.

Abstract

Improvement of disease resistance is one of the most important aims in hop breeding and there is a need for the development of marker assisted resistant breeding. In the present work, a PCR strategy was used to clone resistance gene analogues (RGAs) using degenerative primers designed at the conserved motif of cloned plant NBS–LRR R genes. Fifty-six sequenced PCR clones showed homologies to various R genes deposited in the GenBank database and these clones were further divided into 17 RGA groups. Phylogenetic analysis of hop RGA groups with cloned R genes revealed a separation of hop RGAs into TIR (7 RGA groups with 16 sequences) and non-TIR (10 RGA groups with 40 sequences) NBS–LRR protein classes. The amino acid identity of hop RGAs to various R genes ranged from 41% (TIR–NBS–LRR) to 81% (non-TIR–NBS–LRR). RGA primer sets, designed on 17 reference RGAs, amplified additional RGA sequences from two hop genotypes which served for the development of 11 RGA markers of which eight amplified PCR products of expected sizes also in various hop cultivars, indicating a high conservation level of RGA sequences within the hop genome and two RGA markers segregated in the mapping family. The structure of RGA sequences and similarity to other R gene or R-gene-like sequences is discussed. This is the first report on RGAs in hop and it provides useful data for further breeding manipulations.